1. Field of the Invention
The present invention relates to a method for effectively recovering .alpha.-L-aspartyl-L-phenylalanine methyl ester (hereafter simply referred to as .alpha.-APM) from a solution mixture containing a methyl ester of the L-aspartic acid .beta.-carboxyl residue of .alpha.-L-aspartyl-L-phenylalanine methyl ester (hereafter simply referred to as .alpha.-A(M)PM), the corresponding demethylated esters (.alpha.-APM, .alpha.-A(M)P) and .alpha.-L-aspartyl-L-phenylalanine (hereafter simply referred to as .alpha.-AP)) and an inorganic chloride.
2. Discusson of the Background
.alpha.-APM is a very useful substance to which considerable attention has recently been paid because it is a peptide, low calorie sweetener. .alpha.-APM is a methyl ester derivative of .alpha.-L-aspartyl-L-phenylalanine. Other methyl ester derivatives are also possible. These compounds have the following structures: ##STR1##
The properties of .alpha.-APM in an aqueous solution are such that it is relatively stable at temperatures lower than normal ambient temperatures, but under the severe conditions of high temperatures or in an acidic media, it readily undergoes various undesirable reactions. For example, .alpha.-APM can undergo demethyl-esterification to form .alpha.-AP. It can also undergo an intramolecular addition/elimination with loss of methanol to cyclize and form .alpha.-AP, or it can cyclize intramolecularly to form cyclized .alpha.-L-aspartyl-L-phenylalanine (hereafter simply referred to as DKP). All of these reactions are possible because .alpha.-APM is a methyl esterified 2 amino acid-long peptide.
Upon isolation and purification of .alpha.-APM from a reaction solution on an industrial scale, severe conditions of high temperatures or acidic conditions are often adopted to enhance the efficiency of the system. In such cases, by-production of undesired .alpha.-AP or DKP is unavoidable.
In addition, .alpha.-APM or .alpha.-AP can be methylated with methanol by-product to form .alpha.-A(M)PM or .alpha.-A(M)P. When .alpha.-APM is isolated and purified from reaction solutions, the formation of .alpha.-A(M)PM or .alpha.-A(M)P can occur not only in aqueous solutions but also, on many occasions, in solution mixtures of methanol and water. In this latter case, the formation of .alpha.-A(M)PM or .alpha.-A(M)P is even more accelerated.
Therefore, in producing .alpha.-APM, the process used suffers from the limitation that a part of .alpha.-APM changes to .alpha.-AP, DKP, .alpha.-A(M)PM or .alpha.-A(M)P during isolation and purification steps. This causes a serious reduction in the yield of .alpha.-APM, and results in that large quantities of valuable products are contained in the mother liquor from which .alpha.-APM is separated.
There are various processes for the synthesis of .alpha.-APM. These include a process which comprises reacting N-protected-L-aspartic anhydride and L-phenylalanine methyl ester in an organic solvent and then splitting the protective groups off in a conventional manner (see U.S. Pat. No. 3,786,039). Another process comprises directly reacting L-aspartic anhydride strong acid addition salts and L-phenylalanine methyl ester (Published Examined Japanese patent application No. 14,217/73). Still other such processes exist.
In any of these processes, the reaction solution obtained contains, in addition to .alpha.-APM, impurities such as the by-produced .beta.-form, the unreacted L-phenylalanine and L-aspartic acid used as raw materials or by-products thereof. Therefore, a method for efficiently separating high purity .alpha.-APM in high overall yields is extremely important to the industrial production of .alpha.-APM. A notable such method comprises contacting impure .alpha.-APM with a hydrohalic acid in an aqueous solvent to precipitate the hydrohalic acid salt of .alpha.-APM. The salt is then subjected to solid-liquid separation and neutralization with an alkali to give .alpha.-APM (U.S. Pat. No. 3,798,207). With this method, the mother liquor remaining after the .alpha.-APM is isolated contains large quantities of salts, generally inorganic chlorides because hydrochloric acid is used as the hydrohalic acid because it is economical.
Accordingly, exhausted mother liquors obtained from the isolation and purification of .alpha.-APM from the industrial scale production of .alpha.-APM contain, in addition to .alpha.-APM a mixture of .alpha.-AP, DKP, .alpha.-A(M)PM, .alpha.-A(M)P and large amouts of inorganic chlorides. The inorganic chlorides vary depending upon the alkali used for the neutralization of the .alpha.-APM hydrochloride. Generally these are NaCl, NH.sub.4 Cl, KCl, etc.
Recovery, of .alpha.-APM from such a mother liquor is extremely advantageous in improving the overall industrial production yield of .alpha.-APM. This recovery contributes to great improvements in the yield at the isolation and purification step of .alpha.-APM and to a considerable reduction in .alpha.-APM production costs. However, it has heretofore been difficult to economically recover .alpha.-APM from such mother liquors advantageously because of the the following problems.
In order to improve the yield of the product, one recycles, to earlier steps, the mother liquor from which the product was once separated. This recycling operation enhances the concentration of the product in the mother liquor via evaporation, concentration, etc. One then again precipitates and separates the product from the mother liquor. This procedure can be repeated several times.
When large quantities of salts are contained in the mother liquor however, the concentrations of the salts becomes high and the salts are precipitated as the recycling procedure is repeated. In this case, it is difficult to recover the product in high yield and recycle the mother liquor, especially since it is advantageous to recover the .alpha.-APM product from economic hydrochloric acid solutions. With this system, the solubility of the inorganic chlorides decreases due to the hydrochloric acid present in the system so that it becomes more difficult to recover the product.
Therefore, in cases where salts (such as inorganic chlorides, etc.) are contained in large quantities, the solution must be previously desalted using any means available.
For such desalting operations, various methods are known. (1) For example, a method using ion exchange resins has been used. (2) A method which comprising adsorbing the product to an adsorbent such as a synthetic adsorbent, activated charcoal, etc., breaking the salts present through a non-adsorbed solution and then eluting with a polar solvent such as methanol, etc., has also been used. Other methods include (3) a method utilizing electric dialysis, and (4) a method using an ultrafiltration membrane, etc.
These methods suffer the common problem that large quantities of a secondary raw material is consumed for elution and regeneration of the ion exchange resins, the absorbents or the other desalting means. Complicated operations increase the costs of using systems (1) and (2). When the product has an electrolytic property, desalting is performed at the same time the product is dialyzed. This decreases the yield of (3).
Methods using an ultrafiltration membrane apply generally to products having a molecular weight of several thousands or more. In the case of products having a lower molecular weight, the product also permeates through the membrane so that the yield of the product is markedly reduced. In addition, the desalted solution thus obtained becomes dilute so that considerable energy is required to concentrate the solution to permit recovery of the product from the desalted solution. Therefore, recovery costs increase to the detriment of the economics of the overall system.
Another problem is that it is extremely difficult to crystallize .alpha.-APM directly from an impure system containing .alpha.-A(M)PM, .alpha.-A(M)P .alpha.-AP, DKP, in large quantities. The precipitation of .alpha.-APM crystals is inhibited by these impurities.
Accordingly, methods which comprises contacting such a solution with a strong acid such as HCl, H.sub.2 SO4, etc. at high temperatures have been used to hydrolyze up to the constituent amino acids, fractionally crystallizing L-Phe and L-Asp, recovering each of these amino acids independently and then reusing them as raw materials.
With this method, the treatment required severe conditions of strong acids and high temperatures. This increases costs for equipment to ensure safe operation and resistance to corrosion. In addition, the method involves the consumption of large quantities of acids and alkalis.
There is thus a strongly felt need for an improved method for recovering .alpha.-APM from mother liquors and other industrially produced solutions containing .alpha.-APM together with other process impurities, such as .alpha.-APM derivatives and chloride ions.